The Clostridioides difficile Cysteine-Rich Exosporium Morphogenetic Protein, CdeC, Exhibits Self-Assembly Properties That Lead to Organized Inclusion Bodies in Escherichia coli.

Clostridioides difficile is an obligately anaerobic, spore-forming, Gram-positive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outermost layer of C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in Escherichia coli filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an E. coli strain with a more oxidative environment led to the loss of the lamella-like organization of CdeC IBs. Additionally, dithiothreitol (DTT) treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Deletions in different portions of CdeC did not affect CdeC's ability to aggregate and form oligomers stable under denaturation conditions but affected CdeC's self-assembly properties. Overall, these observations have important implications in further studies elucidating the role of CdeC in the exosporium assembly of C. difficile spores.IMPORTANCE The endospore of Clostridioides difficile is the vehicle for transmission and persistence of the pathogen, and, specifically, the exosporium is the first contact between the host and the spore. The underlying mechanisms that govern exosporium assembly in C. difficile remain understudied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. Understanding the exosporium assembly's molecular bases may be essential to developing new therapies against C. difficile infection.

Evaluation of functionality of type II toxin-antitoxin systems of Clostridioides difficile R20291.

Clostridioides difficile is a nosocomial, Gram-positive, strictly anaerobic, spore-forming pathogen capable of colonizing and proliferating in the human intestine. In bacteria, it has been shown that the Toxin-Antitoxin systems mediate the cellular response to external stress by initiating processes such as biofilm formation and programmed cell death. This work aims to evaluate the functionality of four type II TA modules of Clostridioides difficile R20291. We performed bioinformatic analysis to search for putative TA systems using the TADB platform. Then we performed a heterologous expression assay to evaluate the functionality of these systems. Our results showed that the MazEF and RelBE systems were functional, suggesting that their corresponding toxins possess an endoribonuclease activity. In conclusion, MazEF and RelBE systems of C. difficile R20291 are functional in a heterologous expression system.

Effect of antibiotic treatment on the formation of non-spore Clostridium difficile persister-like cells.

Background: The spore is the virulence factor identified to be involved in the recurrence of the disease caused by Clostridium difficile. Objectives: To demonstrate that lethal antibiotic concentrations induce the appearance of C. difficile persister-like non-spore cells. Methods: C. difficile and derivative spo0A mutant strains were tested for their susceptibility to antibiotics, as determined using an agar dilution method. Persister-cell generation was determined for all strains using up to 10â€Š× the MIC of every antibiotic for up to 6 days. Results: Using up to 10â€Š× the MIC of every antibiotic, we were able to induce the appearance of persister-like behaviour since biphasic killing curves could be observed in response to treatment antibiotics. Conclusions: To the best of our knowledge, this work provides, for the first time, experimental evidence of the appearance of C. difficile persister-like cells, opening a new research avenue in the pathogenesis of this nosocomial pathogen.

Salmonella Typhimurium exhibits fluoroquinolone resistance mediated by the accumulation of the antioxidant molecule H2S in a CysK-dependent manner.

OBJECTIVES: To evaluate the contribution of cysK and cysM to the fluoroquinolone (ofloxacin) antibiotic resistance in Salmonella Typhimurium, and their impact on H2S and cysteine production through targeted mutagenesis. METHODS: Salmonella Typhimurium 14028s and its cysK and cysM mutants were tested for their susceptibility to ofloxacin, as determined by a broth microdilution test (to determine the MIC) and survival curves. H2S levels were measured by the Pb(AC)2 method and cysteine levels were determined using 5,5-dithio-bis-2-nitrobenzoic acid. DNA damage induced by antibiotic treatment was determined by PFGE. Finally, expression of cysK and cysM genes under antibiotic treatment was determined by real-time reverse transcription PCR. RESULTS: As determined by MIC, the ΔcysK strain was more resistant to ofloxacin, a reactive oxygen species (ROS)-producing fluoroquinolone, than the WT and ΔcysM strains, which correlates with survival curves. Moreover, the ΔcysK strain exhibited higher H2S levels and lower cysteine levels than the WT strain. Finally, the ΔcysK strain exhibited lower DNA damage upon challenge with ofloxacin than the WT and ΔcysM strains. These results are in accordance with lower expression of cysK under ofloxacin treatment in the WT strain. CONCLUSIONS: This work demonstrated that cysteine metabolism in Salmonella Typhimurium modulated H2S levels, conferring resistance to second-generation fluoroquinolones.

Lose to win: marT pseudogenization in Salmonella enterica serovar Typhi contributed to the surV-dependent survival to H2O2, and inside human macrophage-like cells.

The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and Salmonella enterica serovar Typhi (S. Typhi) can be partially attributed to the gain of functions, to the loss of functions (i.e. pseudogenization), or to a combination of both processes. As previously reported, the loss of functions by pseudogenization may play a role in bacterial evolution, especially in host-restricted pathogens such as S. Typhi. The marT-fidL operon, located at the SPI-3, encodes the MarT transcriptional regulator and a hypothetical protein (i.e. FidL) with no significant similarities to known proteins, respectively. Even though predicted S. Typhimurium FidL exhibit 99.4% identity with S. Typhi FidL, marT has been annotated as a pseudogene in S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium marT-fidL exhibited an increased accumulation of reactive oxygen species (ROS), leading to a decreased survival in presence of H2O2. Moreover, we found that that the presence of a functional copy of S. Typhimurium marT-fidL in S. Typhi resulted in a repression of surV (STY4039), an ORF found in the S. Typhi SPI-3 but absent from S. Typhimurium SPI-3, that contribute to the resistance to H2O2 by decreasing the accumulation of ROS. Finally, we observed that the presence of S. Typhimurium marT-fidL in S. Typhi negatively affected the survival inside macrophage-like cells, but not in epithelial cells, after 24h post infection. Therefore, this work provides evidence arguing that marT pseudogenization in Salmonella Typhi contributed to the surV-dependent survival against H2O2, and inside human macrophage-like cells. This is a good example of how the loss of functions (marT pseudogenization) and the gain of functions (presence of surV) might contribute to phenotypic changes improving virulence.

Predominance of Clostridium difficile ribotypes 012, 027 and 046 in a university hospital in Chile, 2012.

In a 1-year survey at a university hospital we found that 20·6% (81/392) of patients with antibiotic associated diarrohea where positive for C. difficile. The most common PCR ribotypes were 012 (14·8%), 027 (12·3%), 046 (12·3%) and 014/020 (9·9). The incidence rate was 2·6 cases of C. difficile infection for every 1000 outpatients.

Pseudogenization of sopA and sopE2 is functionally linked and contributes to virulence of Salmonella enterica serovar Typhi.

The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and S. enterica serovar Typhi (S. Typhi) can be partially attributed to pseudogenes. Pseudogenes are genomic segments homologous to functional genes that do not encode functional products due to the presence of genetic defects. S. Typhi lacks several protein effectors implicated in invasion or other important processes necessary for full virulence of S. Typhimurium. SopA and SopE2, effectors that have been lost by pseudogenization in S. Typhi, correspond to an ubiquitin ligase involved in cytokine production by infected cells, and to a guanine exchange factor necessary for invasion of epithelial cells, respectively. We hypothesized that sopA and/or sopE pseudogenization contributed to the virulence of S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium sopE2 exhibited a decreased invasion in different epithelial cell lines compared with S. Typhi WT. S. Typhimurium sopA completely abolished the hypo-invasive phenotype observed in S. Typhi expressing S. Typhimurium sopE2, suggesting that functional SopA and SopE2 participate concertedly in the invasion process. Finally, the expression of S. Typhimurium sopA and/or sopE2 in S. Typhi, determined changes in the secretion of IL-8 and IL-18 in infected epithelial cells.

Effects of wet heat treatment on the germination of individual spores of Clostridium perfringens.

AIM: To analyse the effect of wet heat treatment on nutrient and non-nutrient germination of individual spores of Clostridium perfringens. METHODS AND RESULTS: Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the dynamic germination of individual untreated and wet heat-treated spores of Cl. perfringens with various germinants. When incubated in water at 90-100°C for 10-30 min, more than 90% of spores were inactivated but 50-80% retained their Ca(2+) -dipicolinic acid (CaDPA). The wet heat-treated spores that lost CaDPA exhibited extensive protein denaturation as seen in the 1640-1680 cm(-1) (amide I) and 1230-1340 cm(-1) (amide III) regions of Raman spectra, while spores that retained CaDPA showed partial protein denaturation. Wet heat-treated spores that retained CaDPA germinated with KCl or l-asparagine, but wet heat treatment increased values of T(lag) , ΔT(release) and ΔT(lys) , during which spores initiated release of the majority of their CaDPA after mixing with germinant, released >90% of their CaDPA and completed the decrease in their DIC intensity because of cortex hydrolysis, respectively. Untreated Cl. perfringens spores lacking the essential cortex-lytic enzyme (CLE), SleC, exhibited longer T(lag) and ΔT(release) values during KCl germination than wild-type spores and germinated poorly with CaDPA. Wet heat-treated wild-type spores germinating with CaDPA or dodecylamine exhibited increased T(lag) , ΔT(release) and ΔT(lys) values, as did wet heat-treated sleC spores germinating with dodecylamine. CONCLUSIONS: (i) Some proteins important in Cl. perfringens spore germination are damaged by wet heat treatment; (ii) the CLE SleC or the serine protease CspB that activates SleC might be germination proteins damaged by wet heat; and (iii) the CaDPA release process seems likely to be damaged by wet heat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the germination of individual Cl. perfringens spores and improves the understanding of effects of wet heat treatment on spores.

Analysis of the germination of individual Clostridium perfringens spores and its heterogeneity.

AIMS: To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens. METHODS AND RESULTS: Germination of individual wild-type Cl. perfringens spores was followed by monitoring Ca-dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (T(lag)) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔT(release)) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex-lytic enzyme (CLE) (sleC spores) exhibited the same features during GR-dependent germination, but with longer average T(lag) values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The T(lag) of wild-type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild-type and sleC spores had identical average T(lag) and ΔT(release) values in dodecylamine germination that does not utilize GRs. CONCLUSIONS: Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE-deficient Bacillus spores, indicating that germination of these species' spores is not completely identical. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.

Combined effects of hydrostatic pressure, temperature, and pH on the inactivation of spores of Clostridium perfringens type A and Clostridium sporogenes in buffer solutions.

To develop a spore inactivation strategy, the effect of 15-min hydrostatic pressure treatments (550 and 650 MPa) at 55 and 75 degrees C in citric acid buffer (4.75 and 6.5 pH) on spores of 5 isolates of Clostridium perfringens type A carrying the gene that encodes the C. perfringens enterotoxin (cpe) on the chromosome (C-cpe), 4 isolates carrying the cpe gene on a plasmid (P-cpe), and 2 strains of C. sporogenes were investigated. Treatments at 650 MPa, 75 degrees C and pH 6.5 were moderately effective against spores of P-cpe (approximately 3.7 decimal reduction, DR) and C. sporogenes (approximately 2.1 DR) but not for C-cpe (approximately 1.0 DR) spores. Treatments at pH 4.75 were moderately effective against spores of P-cpe (approximately 3.2 DR) and C. sporogenes (approximately 2.5 DR) but not of C-cpe (approximately 1.2 DR) when combined with 550 MPa at 75 degrees C. However, when pressure was raised to 650 MPa under the same conditions, high inactivation of P-cpe (approximately 5.1 DR) and C. sporogenes (approximately 5.8 DR) spores and moderate inactivation of C-cpe (approximately 2.8 DR) spores were observed. Further advances in high-pressure treatment strategies to inactivate spores of cpe-positive C. perfringens type A and C. sporogenes more efficiently are needed.